Overexpression of Mitochondrial Hsp70/Hsp75 Protects Astrocytes against Ischemic Injury in vitro

Astrocyte Culture

Primary astrocyte cultures were prepared as described previously (Dugan et al, 1995) in accordance with a protocol approved by the Stanford University Animal Care and Use Committee. Briefly, primary astrocyte cultures were prepared from postnatal (days 1 to 3) Swiss Webster mice (Simonsen, Gilroy, CA, USA). The cortices were dissected and treated with 0.05% trypsin for 15mins, mechanically dissociated, and suspended in plating medium consisting of MEM (minimal essential medium) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), 10% equine serum (Hyclone), 2mmol/L glutamine, and 10ng/mL epidermal growth factor (Sigma, St Louis, MO, USA). They were plated as a single-cell suspension at a density of two hemispheres per 24-well plate. Cultures were maintained at 37°C in a 5% CO₂ incubator. After 3 days, half the medium was removed and replaced with astrocyte growth medium (MEM supplemented with 10% equine serum and 2mmol/L glutamine) and this was repeated every 3 days. Experiments were performed after 21 to 40 days in culture. We observed previously that astrocyte vulnerability to injury increased until approximately 21 to 25 days in culture (Papadopoulos et al, 1998), and was then relatively stable through 60 days in culture (Xu et al, 2004).

Viral Construct and Production

The human Hsp75 coding sequence (GenBank L15189) in pBluescript (Bhattacharyya et al, 1995) was a generous gift from R Morimoto. The DNA was cut with SalI, the overhang was blunted using T4 DNA polymerase, and a fragment excised with BamHI, yielding the entire Hsp75 coding sequence plus 692 bases of 3′-untranslated region. We cloned this into HpaI-BamHI cut LXSN vector (Miller and Rosman, 1989) to create LXSN-Hsp75. To produce virus, LXSN-Hsp75 or control empty vector LXSN DNA was transfected into the Ψ−2 ecotropic packaging cell line (Mann et al, 1983), using FuGENE-6 (Roche, Pleasanton, CA, USA). Culture supernatants were harvested 48 to 72 h posttransfection and filtered through 45-micron filters before use to transduce astrocyte cultures.

Hsp75 Overexpression

For Hsp75 or control vector, viral transduction sister cultures in the same 24-well plate were used with 4 to 8 replicates each of (1) control nontransduced, (2) Hsp75-transduced, and (3) control vector-transduced astrocytes. Transduced cultures were treated with packaging cell supernatants of either Hsp75 or control virus mixed 1:1 with plating medium and containing 8 μg/mL polybrene (Sigma) for 24 h, 48 to 72 h after plating. Transduction was repeated twice. Successfully transduced astrocytes were selected in 400 μg/mL G418 (Sigma) for 5 days. The viral transduction did not cause any significant astrocyte death. The antibiotic selection process killed nontransduced astrocytes, so experiments were performed on confluent cultures of cells in which essentially all cells stably expressed Hsp75, or contained control vector, approximately 3 weeks after transduction. The transduced cultures were fed every 3 days with astrocyte growth medium supplemented with 10ng/mL epidermal growth factor for the first 2 weeks after the antibiotic selection. After the 2-week period, half the medium was removed every 3 days and replaced with astrocyte growth medium. Glucose deprivation or oxygen-glucose deprivation (OGD) used the same protocol for transduced and nontransduced cultures.

Western Blot Analysis

Equal amounts of protein as determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA), 30 μg per condition, were separated on a 12.5% Polyacrylamide gel and electrotransferred onto Immobilon polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked with 5% nonfat dry milk in water with 0.05% Tween 20 (blocking buffer) for 1h, incubated overnight with 1:1,000 dilution of primary anti-Hsp75 antibody (Stressgen, Ann Arbor, MI, USA) in blocking buffer, washed three times with 0.05% Tween in phosphate-buffered saline (PBS), and incubated with 1:2,000 anti-mouse HRP (horseradish peroxidase)-linked antibody (Cell Signaling, Danvers, MA, USA) in blocking buffer for 90 mins. Immunoreactive bands were visualized with the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA) according to manufacturer's protocol. The membranes used for Hsp75 detection were reprobed with 1:1,000 anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The Hsp75 band intensity was normalized to the actin band intensity in the same lane.

Glucose Deprivation

All cells had their medium changed to BSS_5.5 containing the following (in mmol/L): glucose, 5.5; NaCl, 116; CaCl₂, 1.8; MgSO₄, 0.8; KCl, 5.4; NaH₂PO₄, 1; NaHCO₃, 14.7, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 (pH 7.4) 24 h before the start of the experiment. Control cells had their medium changed to BSS₅₅, whereas GD was induced by completely exchanging the medium for BSS_o, which was identical to BSS_5.5 but lacking glucose. BSS_o was added to each well to bring the total volume to 1 mL. This was followed by three washes in which 0.8 mL medium was removed and replaced by 0.8 mL BSS_o. After the final wash, the volume was adjusted to 0.5 mL.

Oxygen-Glucose Deprivation

Cultures were transferred to an anaerobic chamber (Coy, Glass Lake, MI, USA) with an atmosphere of 5% CO₂, 10% H₂ and 85% N₂. The culture medium was replaced three times with deoxygenated BSS_o using the same washing scheme described in the GD method above. Cultures were then placed in a humidified 37°C incubator within the anaerobic chamber. Oxygen tension was monitored with an oxygen electrode (Microelectrodes, Bedford, NH, USA) and was kept under 0.02%. Oxygen-glucose deprivation was ended by adding glucose to the culture medium to a final concentration of 5.5 mmol/L and returning the cultures to the normoxic incubator for 24 h before cell death evaluation. Experimental data were combined from different experiments in which OGD caused ˜30% cell death in control vector-transduced astrocytes. Depending on the cell isolation, cells from different dissections required 4.5 to 5.5 h OGD followed by 24 h reperfusion to attain this level of injury.

Live Imaging

The live imaging studies were performed as described previously (Ouyang et al, 2006). Briefly, oxygen radical production was monitored using hydroethidine (HEt). Cultures were loaded in the dark with 5 μmol/L HEt for 30mins at 37°C, and the same concentration of HEt was maintained in the bath throughout each experiment. Cells were excited at 495 nm, and fluorescence emission monitored at 530 nm. Fluorescence measurements for each cell (F_t) were normalized to the initial fluorescence intensity (F_o) for that cell. For assessment of changes in mitochondrial membrane potential, astrocytes were incubated for 30mins with tetramethylrhodamine ethyl ester (TMRE) (50nmol/L) at 37°C, and the same concentration of TMRE was maintained in all bathing solutions throughout the experiments. Cells were excited at 535 nm, and fluorescence emission was monitored at 590 nm. Changes in fluorescence were quantified by selecting mitochondria-rich perinuclear regions in each cell, and all subsequent fluorescence measurements were normalized to the baseline fluorescence (F_o) for the same cell at the start of the experiment.

Immunocytochemistry

Fluorescence immunocytochemistry was performed on cell cultures in 24-well plates. The wells were washed twice in PBS and then fixed in 4% paraformaldehyde for 30mins at room temperature. The cells were then washed twice with PBS and then in PBS containing 0.3% Triton X-100 for 15 mins. Nonspecific binding sites were blocked in blocking buffer (3% bovine serum albumin and 0.3% Triton X-100 in PBS) for 1 h. The cells were incubated with anti-Hsp75 primary antibody (Stressgen) at 1:300 dilution in blocking buffer overnight at 4°C and then washed three times with blocking buffer, 10 mins per wash. The cells were incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) at 1:100 dilution in blocking buffer at RT for 1 h, then washed 2 times in blocking buffer, and one time in PBS, 10 mins per wash. Fluorescence was visualized with an epifluorescence microscope (Zeiss Axiovert 200mol/L; Carl Zeiss, Göttingen, Germany), and images were obtained on a Macintosh computer using Openlab software from Improvision Inc. (Lexington, MA, USA).

ATP Measurements

Astrocyte intracellular ATP levels were quantitated immediately at the end of 4.5 h of OGD exposure. Cellular ATP concentrations were measured using the CellTiter-Glo luminescent ATP assay kit, based on the luciferase/luciferin reaction (Promega, Madison, WI, USA) according to the manufacturer's instructions. A Veritas luminescence counter (Turner BioSystems, Sunnyvale, CA, USA) was used to measure the luminescence signal of the samples in opaque white 96-well plates. ATP standards (Amersham) were used to create a calibration curve. Protein concentrations were measured using a bicinchoninic acid protein assay from Pierce per the manufacturer's instructions.

Cell Viability Assays

Cell death was quantified by microscopic evaluation of Hoechst 33342 (5 μmol/L) and propidium iodide (PI, 5 μmol/L)-labeled cells. PI readily penetrates cells with compromised plasma membranes (dead cells) but does not cross intact plasma membranes. Hoechst is a cell-permeant nucleic acid stain that labels both live and dead nuclei.

Statistics

Statistical differences between two groups were determined using unpaired two-tailed Student's t-test. Comparisons between multiple groups were performed with analysis of variance followed by Tukey's multiple comparison test, with the P <0.05 level considered significant. Data in all plots are presented as mean ± s.d.

Overexpression of Hsp75

Previous studies have revealed endogenous expression of Hsp75 in various tissues including normal and ischemic brain tissue (Massa et al, 1995). Retroviral transduction of primary astrocyte cultures with Hsp75 resulted in markedly higher levels of Hsp75 expression compared to those in vector-transduced astrocytes and to endogenous Hsp75 levels in control cells, as indicated by Western blot (Figure 1A). Hsp75 overexpression in LXSN-Hsp75-transduced astrocytes, along with preferential mitochondrial localization, was also confirmed by immunocytochemistry (Figure 1B).

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Figure 1

Vector-transduced cells have higher levels of Hsp75. (A) Expression levels of Hsp75 in uninfected control, vector-transduced, and Hsp75-transduced astrocytes as determined by Western blot (*P < 0.05 compared to vector-transduced astrocytes). (B) Immunohistochemical Hsp75 staining of vector- and Hsp75-transduced astrocytes shows typical mitochondrial punctuate perinuclear fluorescence. Scale bar, 25 μm.

Protection from Glucose Deprivation

We previously showed that GD induced increased levels of oxidative stress in primary astrocyte cultures (Ouyang et al, 2006; Papadopoulos et al, 1997). Intracellular ROS levels were evaluated using the ROS-sensitive fluorescent dye HEt (Figure 2). We found that HEt staining was not significantly different between the different groups of astrocytes at Oh GD. Upon exposure to GD, uninfected and vector-transduced astrocytes have shown immediate and rapid ROS generation, with HEt fluorescence increasing by six-fold in both groups after 2 h of GD. In contrast, Hsp-75-overexpressing astrocytes showed more moderate increases in ROS levels, with the HEt signal increasing approximately two-fold after 2 h of GD.

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Figure 2

Hsp75 overexpression reduces ROS accumulation induced by glucose deprivation (GD) (A) Examples of changes in hydroethidine (HEt) fluorescence before (t = 0, left column) and after 2 h of GD (right column). Scale bar, 50μm. (B) Time course of ROS accumulation with GD in uninfected control, vector-transduced, and Hsp75-overexpressing astrocytes. For each cell, the (HEt) fluorescence was normalized to the starting fluorescence for that cell. The traces represent mean ± s.d. of at least 50 astrocytes per condition, derived from at least three experiments (*P<0.05 compared to both control and vector-transduced under the same condition).

Mitochondria are both targets and sources of cellular oxidative stress (Fiskum et al, 2004). We have also previously reported that GD promoted mitochondrial depolarization in primary astrocyte cultures (Ouyang et al, 2006). Mitochondrial functions including protein import and generation of ATP are dependent on maintenance of the mitochondrial membrane potential (Voisine et al, 1999). To investigate the effect of Hsp75 overexpression on changes in mitochondrial membrane potential induced by GD, we used the fluorescent dye TMRE, which accumulates in mitochondria in a potential-dependent manner. As indicated in Figure 3, at Oh of GD, all groups showed similar levels of TMRE fluorescence with a typical mitochondrial pattern of perinuclear staining. After 2 h of GD, the TMRE signal of Hsp75-overexpressing astrocytes did not significantly change compared to the Oh time point. In contrast, TMRE signal in control and vector-transduced astrocytes was reduced by ˜15% (P<0.05) indicating partial mitochondrial depolarization.

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Figure 3

Hsp75 overexpression preserves mitochondrial membrane potential during glucose deprivation (GD). (A) Examples of TMRE staining before (t = 0, left column) and after 2 h of GD (right column). Scale bar, 50μm. (B) Changes in TMRE mitochondrial fluorescence with GD in uninfected control, vector-transduced, and Hsp75-overexpressing astrocytes. The data are representative of mean ± s.d. of three independent experiments, with at least 50 astrocytes per condition (*P < 0.05 compared to both control and vector-transduced under the same condition).

Protection from Oxygen-Glucose Deprivation

We next investigated whether Hsp75 overexpression protects astrocytes against OGD. ATP depletion is the ultimate consequence of glucose depletion and inhibition of mitochondrial oxidative respiration due to anoxia under OGD conditions. Intracellular ATP levels were quantitated at the end of 4.5 h of OGD exposure. As shown in Figure 4, OGD treatment resulted in similar decreases of 66 and 62% in uninfected and empty vector-transduced astrocytes, respectively. The same OGD exposure caused a 32% decrease in ATP levels of Hsp75-overexpressing astrocytes, significantly less than either control. To investigate the effect of Hsp75 overexpression on astrocyte viability, the extent of astrocyte death was evaluated using PI staining. As shown in Figure 5, the same OGD exposures induced approximately 25% astrocyte death in uninfected and control-transduced cultures, but caused only 5% cell death in Hsp75-overexpressing astrocytes.

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Figure 4

Hsp75 overexpression partially preserves ATP levels in astrocytes subjected to 4.5 h of OGD. The data are representative of mean ± s.d. of three independent experiments, each performed in duplicate (*P<0.05 compared to both control and vector-transduced under the same condition).

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Figure 5

Hsp75 overexpression increases survival of astrocytes subjected to oxygen-glucose deprivation (OGD) and 24h of recovery. Experimental data from three experiments in which OGD for 4.5 to 5.5 h achieved ˜30% cell death in control vector-transduced astrocytes were pooled. Cell death was significantly decreased in Hsp75-overexpressing astrocytes (A) compared to vector-transduced (B) and uninfected control (C), as assessed by PI staining. Scale bar, 50μm. (D) Quantification of OGD-induced astrocyte death, mean ± s.d. of three independent experiments, 400 to 500 cells for each condition in each experiment (*P<0.05 compared to both control and vector-transduced under the same condition).